Advancing age is the primary risk factor for cardiovascular diseases (CVD) due largely to adverse changes to arteries. A major feature of arterial aging is the development of vascular endothelial dysfunction as assessed by a decline in endothelium-dependent dilation (EDD). Impaired EDD with advancing age is caused by reduced bioavailability of the vasodilatory and vascular protective molecule nitric oxide (NO), secondary to oxidative stress. A major contributor to age-associated vascular oxidative stress is excessive production of mitochondrial reactive oxygen species (mtROS). As such, therapies that reduce mtROS may improve vascular endothelial function and decrease CVD risk in middle-aged/older (MA/O) adults. Mitoquinone (MitoQ) is a mitochondria-targeted antioxidant that, in contrast to traditional exogenous antioxidants, accumulates at levels 100-1,000-fold higher in the mitochondria vs. cytosol of cells, and reduces mtROS-mediated oxidative stress in vivo. Preclinical findings from our lab indicate that 4 weeks of MitoQ supplementation in the drinking water completely restores NO-mediated EDD in old (26-28 mo) mice, and reverses the age-related increases in aortic mitochondrial superoxide production and oxidative stress. The goal of the current study is to translate our preclinical findings to MA/O adults. We propose a randomized, double-blind, placebo-controlled cross-over pilot study to provide the first evidence for the efficacy of oral MitoQ supplementation to improve NO-mediated EDD in healthy MA/O adults (60-79 yr; n=21) with baseline endothelial dysfunction. To gain insight into the mechanisms by which MitoQ may improve EDD in this group, we will employ innovative translational techniques to assess endothelial cell mtROS production, mtROS-mediated suppression of EDD, and systemic and/or vascular endothelial cell markers of mitochondrial function, oxidative stress, and inflammation. Hypothesis 1: 6 weeks of oral MitoQ supplementation will increase NO-mediated EDD in MA/O adults. Hypothesis 2: Improved EDD with MitoQ treatment will be mediated by reduced endothelial cell mtROS production and mtROS-related suppression of EDD, and will be associated with decreased vascular endothelial and systemic oxidative stress. This investigation will combine clinically relevant functional assessments with innovative mechanistic analyses to investigate a novel intervention approach for reducing mitochondria-derived oxidative stress and improving NO-mediated EDD in MA/O adults with endothelial dysfunction, but free of clinical disease.